Starburst amacrine cells (SACs) are radially symmetric inhibitory interneurons essential to confer the direction- selective (DS) properties of DS visual circuits in the mammalian retina, which impart image motion detection, a critical function for organismal survival. SACs have evolved to release neurotransmitter from varicosities in their distal dendritic arbors, since they lack axons. The principles that govern circuit assembly of neurons that lack clearly distinguishable axonal-dendritic compartments, and therefore discrete regions of specialization, are poorly defined. In the mouse retina, signaling through the guidance cue semaphorin6A (Sema6A) and its receptor is required for proper SAC circuit assembly. Two mirror populations of SACs exist that respond to either increases (ON) or decreases (OFF) in light. SAC are initially intermingled in the developing retina, but then stratif into the ON and OFF SAC circuits. Sema6A-PlexA2 signaling drives not only ON and OFF SAC stratification, but also establishes appropriate radial symmetry of individual ON SACs, which is necessary for accurate DS visual responses. Sema6A is also required for proper light (i.e. visual cue) evoked inhibitory postsynaptic responses in ON SACs. How the Sema6A-PlexA2 signaling pathway elicits such disparate cellular responses is unclear. This proposal is designed to study the mechanisms that distinguish these branches of guidance cue signaling within single SACs, and to define the range of guidance cues that contribute to DS SAC wiring. The specific aims of this study are to 1) assess the directionality of Sema6A-PlexA2 signaling pathways during retinal development with respect to segregation of the ON and OFF SAC circuits and development of SAC symmetry; 2) probe the mechanism underlying improper SAC inhibition in Sema6A mutants; and 3) to ask if other class 6 Semas and/or class A Plexins contribute to wiring DS circuitry. I will address these basic biological questions by 1) conditional removal of either Sema6A or PlexA2 in single SACs to determine the directionality of signaling; 2) examining the localization of synapses in Sema6A and PlexA2 mutant SACs, to ask if inappropriate synapse formation contributes to improper SAC inhibitory input; and 3) phenotypic analysis of class 6 Sema and class A double mutant combinations, to ask if any of these molecules regulates SAC stratification or elaboration of SAC symmetry. The answers to these questions have the potential to reveal general principles that guide CNS circuit assembly, especially in contexts where neurons lack axodendritic polarity. Since these molecules also contribute to wiring other circuits in the developing CNS, in addition to wiring DS circuits in the retina, understanding the directionality of their signaling and the mechanisms by which they direct distinct aspects of circuit refinement in the retina will have significant implications for treating developmental neuronal circuitry disorders where these and related signaling molecules are non-functional.